Mammalian phosphatidylinositol specific phospholipase C (PI-PLC) enzymes are multidomain proteins that are key regulators of phosphoinositide-signaling, They have critical roles in cell growth and proliferation. The bacterial PI-PLC enzymes, single domain proteins structurally equivalent to the catalytic domain of the mammalian enzymes, are usually secreted and often aid in the infection of target cells. Although the three proteins we study, Bacillus thuringiensis PI-PLC, Listeria monocytogenes PI-PLC, and mammalian PLC41, share a common structural framework for the catalytic domain, their interaction with and regulation by lipids is different and tied up with their individual physiological functions. Our long-term goal is to derive a molecular level picture of how these proteins dock on their target bilayer membranes. Specifically, we plan to (i) define and characterize specific nonsubstrate/allosteric phospholipid binding site(s) using high resolution field cycling 31P NMR relaxation experiments; (ii) orient these PI-PLCs on model membranes, particularly with respect to the mobile loops and hydrophobic ridge residues surrounding the active sites using NMR and HXMS methods; and (iii) extend PI-PLC binding studies, using FCS and single molecule fluorescence microscopy, from simple vesicles to more complex target cell-like membranes. The results from this work should show how these enzymes interact with different components of the membrane surface and how binding at nonsubstrate interfacial sites is translated to enhanced catalysis. L. monocytogenes is an intracellular pathogen of humans that can cause serious infections in immunocompromised individuals. Insight into how this PI-PLC, which contributes to virulence, interacts with intracellular membranes may provide alternate ways of slowing intracellular growth of this organism. Likewise some Bacillus cereus strains are opportunistic pathogens and PI-PLC cleavage of GPI-anchored proteins is likely to contribute to virulence. For PLC41, the emphasis is on autoinhibition of the enzyme by a disordered peptide region that may be an important way of controlling basal activity of the enzyme.